Vincenzo Di Marzo Endocannabinoid id Research Group C di t d b Istituto di Chimica Biomolecolare C.N.R., Pozzuoli (NA), Italy

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1 Effects of Krill Oil Supplementation on the Endocannabinoid System Vincenzo Di Marzo Endocannabinoid id Research Group C di t d b Istituto di Chimica Biomolecolare C.N.R., Pozzuoli (NA), Italy Presented at Coordinated by Rejuvenation Science A4M 2011 Orlando, FL Brief introduction to the endocannabinoid (ecb) system and its role in the adipose tissue and liver Dysregulation of the ecb system in adipose depots, skeletal muscle and liver in abdominal obesity/hyperglycemia, and its consequences Causal relationship between ecb dysregulation and dysmetabolism Omega-3 PUFAs and krill oil against ecb system dysregulation

2 Krill oil is an efficient utilization of the world s largest biomass Antarctic Krill largest biomass on earth Efficient utilization of nature s resources Antarctic Krill (Euphausia superba) World s largest single species biomass of mill MT (almost (l double size of human beings) Live in large schools; up to 6 years Feed directly on phytoplankton p Pelagic life cycle Output comparison: 10 times more output < Fish Oil Krill Oil

3 Cannabinoid receptors CH 3 OH O OH O CH O (-)- 9 -tetrahydrocannabinol (THC, oil) 1 M L 1 OH 2-Arachidonoyl-glycerol Hippocampus Basal Ganglia Cortex Cerebellum Hypothalamus Limbic structures Brainstem G.I. Tract Adipocytes CB 1, CB 2 E Immune cells and tissues Adipocytes?

4 Endocannabinoids (ecbs) (endogenous agonists of cannabinoid receptors) 1) are produced on demand 2) activate cannabinoid receptors locally and exert a prohomeostatic function 3) are metabolized thereafter Phospholipid Remodelling R 1 O Phospholipid-derived precursorsrsors NAPE-PLD O N H O O P O O- O-R 2 O O-R 3 O CH OH DAG Lipase Endocannabinoids O N H OH O O OH CH OH Anandamide 2-Arachidonoyl-glycerol Fatty Acid Amide Hydrolase MAG Lipase Degradation products H 2 N OH O OH OH HO CH OH

5 CB 1 receptors play a crucial role in adipogenesis and lipogenesis Di Marzo, Diabetologia 2008 CB 1 receptor activation also reduces adipocyte mitochondrial biogenesis (Tedesco et al., Diabetes, 2010)

6 Dysregulation of the Adipose Tissue, Hepatic and Skeletal Muscle ecb System in Obesity and Type 2 Diabetes

7 Amounts Aberrant ecb system in the epididymal and subcutaneous adipose tissue of DIO mice epidydimal fat control diet diet-induced obesity * Amounts 60,0 40,0 20,0 2,0 1,5 ** Lean DIO 1 1,0 0 anandamide pmol/g damide (pmol/g wet tissue) 60 2-AG nmol/g ad lib fasted refed 0,5 0,0 Anandamide (pmol/g) ** 2-AG (nmol/g) Matias et al. J. Clin. Endocrinol. Metab Starowicz et al., Obesity, subcutaneous ad lib mesenteric fasted refed * Anan Lean ### **, ## Zucker #,# Izzo, Piscitelli, Capasso, Aviello,, Romano, Borrelli, Petrosino,, Di Marzo, 2009,## Zucker

8 Elevated ecb tone in human visceral adiposity and metabolic syndrome Endogenous ligands (ecbs) Visceral fat from normoweight patients (n=10) Visceral fat from obese patients (n=19) Subcutaneous fat from obese patients (n=8) ** CB 1 receptors pmol/g of tiss sue # # 20 0 Matias et al. J. Clin. Endocrinol. Metab Anandamide 2-AG (Sarzani et al., Metabolism, 2009)

9 The ecb system is down-regulated in the subcutaneous adipose tissue of obese/ow/t2d subjects CB 1 receptors CB 1 receptors Endogenous ligand (2-AG) (Bennetzen et al., Eur. J. Clin. Invest., 2009) (Sarzani et al., Metabolism, 2009) (Annuzzi et al., 2010)

10 ecb dysregulation might contribute to fat redistribution and ectopic (liver) fat formation Hypertrophic subcutaneous adipocytes from obese individuals Less and less fat in subcutaneous depots EC Adipocytes from lean individuals EC Hypertrophic visceral adipocytes from obese individuals More and more fat in visceral depots and possibly the liver

11 Elevated CB 1 tone in the liver of obese rodents DIO mice Zucker fa/fa rats AEA (fmol/m mg tissue) p<0.005 Standard diet Anandamide (p pmol/g wet tissue) 40 *** Lean *, ad lib fasted refed # ** Zucker 2-A AG (pmol/mg g/tissue) High fat diet 2-AG (nmol/g wet tis ssue) ** ** ad lib fasted refed ## Osei-Hyiaman et al J Clin Invest Lean Zucker Izzo et al Br. J. Pharmacol 2009

12 Liver-specific CB 1 null mice: hepatic CB 1 receptor is partly responsible for the development of high fat diet-inducedinduced fatty liver, dyslipidemia and insulin resistance Osei-Hyiaman et al J Clin Invest 2008

13 CB 1 Antagonism in the Skeletal Muscle Enhances Insulin Sensitivity and Glucose Uptake The cannabinoid CB 1 receptor antagonist rimonabant stimulates 2- deoxyglucose uptake in skeletal muscle cells by regulating the expression of phosphatidylinositol-3-kinase (Esposito et al., Mol Pharmacol. 2008) Cannabinoid type 1 receptors in human skeletal l muscle cells participate i t in the negative crosstalk between fat and muscle (Eckardt et al. Diabetologia. 2009) 2-AG (nmol/g) High fat diet Regulation of MAP kinase-directed mitogenic and protein kinase B- mediated signaling by cannabinoid receptor type 1inskeletal muscle cells (Lipina et al., Diabetes 2010) Matias et al. Mol. Cell. Endocrinol Effects of in vitro antagonism of endocannabinoid-1 receptors on the glucose transport system in normal and insulin-resistant rat skeletal muscle (Lindborg et al. Diabetes Obes Metab. 2010) Normal diet

14 Peripheral overactivity of the endocannabinoid system 1) strictly correlates with increased visceral AT, liver fat and insulin resistance

15 Correlations between plasma 2-AG levels and cardio- metabolic risk factors in abdominal obese male subjects Variables 2-AG AEA Bd Body mass index id (kg/m (k/ ) r=0.30, 030 r=-0.13, 013 p< NS Waist circumference (cm) r=0.31, r=-0.19, p<0.02 NS Intra-abdominal AT (cm 2 ) r=0.45, r=-0.37, p< p<0.003 Subcutaneous AT (cm 2 ) r=0.07, r=-0.11, NS NS HDL cholesterol (mmol/l) r=-0.25, r=0.16, p<0.04 NS Triglycerides (mmol/l) r=0.35, r=-0.20, p<0.005 NS Fasting insulin (pmol/l) r=0.35, r=-0.26, p<0.005 p<0.04 Fasting glucose (mmol/l) r=0.16, r=-0.01, NS NS Insulin area (pmol/lx10-3 ) r=0.35, r=-0.15,,p<0.006 NS Glucose area (mmol/lx10-3 ) r=0.36, r=-0.04, p<0.005 NS Adiponectin ( g/ml) r=-0.30, r=0.18, p<0.02 NS Côté, Matias, Lemieux, Petrosino, Alméras, Després, Di Marzo, Int. J. Obes., 2007

16 One year lifestyle modification (healthy eating and physical activity) inducing a 8 cm reduction in waist circumference reduces plasma ecb levels in abdominally obese male patients 0,0 Di Marzo, Côté, Matias, Lemieux, Arsenault, Cartier, ndiabetologia, Piscitelli, Petrosino, Alméras & Després, g, 2009 wing intervention ol/ml) Changes follow (pmo -0,5-1,0 *** Anandamide **** 2-AG Positive correlations between 2-AG levels with: visceral adipose tissue (VAT); Free TG and TG in HDL, LDL and VLDL cholesterol; HDL 3 -cholesterol; insulin resistance Multivariate analyses showed that: The decrease in 2-AG and the decrease in VAT explained 13.7% and 38.0% of the decrease in TG, respectively; 20.3% of the decrease in RepIns30 was explained by the decrease of 2-AG

17 Peripheral overactivity of the endocannabinoid system 2) concurs to increased visceral AT, liver fat and insulin resistance

18 Pharmacologically- or genetically-inducedinduced ecb overactivity in mice leads to high triglycerides in a CB 1 -dependent way IDFP= non-selective inhibitor of ecb enzymatic hydrolysis that increases tissue ecb levels Ruby et al, PNAS U.S.A., 2008 FAAH null mice under a high fat diet. Tourino et al, Int. J. Obes., 2010

19 Weight-loss-Independent Effects of Rimonabant in Clinical Trials One year treatment with rimonabant (20 mg, per os) Placebo subtracted effects Di Marzo, Diabetologia, 2008

20 CB 1 antagonism reduces visceral adiposity in candy-diet diet fed female rats and obese humans Visceral fat Herling et al., Int. J. Obes Subcut. fat Strong reduction of liver fat too The ADAGIO-Lipids study Després et al., ATVB, 2009

21 Global CB 1 receptor antagonists are no longer in clinical development: what s next? Rimonabant Cl Cl Taranabant O F F N NH N N NH O N F Cl Cl N O N N Cl N N N NH O NH 2 Cl Otenabant

22 Future strategies for ecb overactivity counteraction Dietary -3 fatty acids against ectopic fat? Four week administration in Zucker fa/fa rats No effect on ecb levels in the subcutaneous fat The effects were stronger with krill oil (KO) than fish oil (FO) and were accompanied by reduction of ecb phospholipid h id biosynthetic precursors There was no effect on food intake and body weight Batetta et al., J Nutr 2009

23 Preventive effect of dietary krill oil in DIO mice (8 weeks co-administration with HFD) Reduction of fasting glycemia with no effect on food intake and body weight Tandy et al., J Agric Food Chem. 2009

24 The effect of dietary krill oil in DIO mice is accompanied by reductions in adipose EC levels Epididymal adipose tissue unpublished Inguinal adipose tissue

25 The effect of dietary krill oil in DIO mice is accompanied by reductions in muscle EC levels # # ** ** ** ** *** *** # # * ** * * *** *** Gastrocnemius muscle unpublished Heart

26 The effect of dietary krill oil in DIO mice is accompanied by reduction in EC precursors unpublished

27 Dietary krill oil lowers (4 weeks) plasma 2-AG levels in human obese subjects (BMI~30, mostly women, N=19-23) nm AG KO B 250 pre 200 post * 50 nm AG OO 150 pre post F Banni et al., Nutr. Metab norm OW OB norm OW OB 7.0 plasma n 6/n n R² = AG (nm)

28 Prolonged dietary krill oil (24 weeks) lowers plasma anandamide and TG levels in obese males (BMI~32, N=10) Plasma data % change Body data % change P<0.05 P<0.05 P<0.05 2,000 1,000 0, Adipon n. O 3 i EP PA DH HA DP PA 3 Omega Cho ol LD DL HD DL TA AG Glucos se P<0.05 in Insuli Week 12 1,000 Week 24 2,000 3,000 4,000 5,000 Week 12 Week 24 Endocannabinoids % change AG AEA PEA OEA Week 12 Week P<0.05 unpublished

29 Dietary -3 fatty acids reduce ecb levels in the brain less than peripheral tissues: less central side effects than CB 1 antagonists? Effect of 4 weeks administration of krill oil (KO) vs. fish oil (FO) No effect on anandamide levels (Di Marzo et al., Int Dairy J, 2010) Two weeks of DHA-supplemented diet in lean mice Woods et al., J Lipid Res, 2010

30 Acknowledgements Gianfranca Carta, Elisabetta Murru,, Lina Cordeddu, Sebastiano Banni Dipartimento di Biologia Sperimentale, Università di Cagliari, Italy Kjetil Berge, Hogne Vik Aker BioMarine, ASA, Oslo, Norway Sally Tandy, Jeffrey S. Cohn Heart Research Institute, Sydney, y, Australia Mikko Griinari Clanet Oy, Helsinki, Finland. ERG Tiziana Bisogno, Luigia Cristino, Luciano De Petrocellis, Roberta Imperatore, Alessia Ligresti, Aniello Schiano Moriello, Pierangelo Orlando, Stefania Petrosino, Fabiana Piscitelli, Noriko Shinjyo, Cris Silvestri, Marco Allarà, Roberta Verde Pozzuoli s ERG